JPH0461638B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating 3-hydroxybutyrate polymers from microorganisms. Poly(3-hydroxybutyrates) are effective against many microorganisms, especially Alcaligenes, Athiorhodium, Azotobacter, Bacillus, Nocardia, Pseudomonas, Rhizobium. (Rhizobium) and Spirillium (Spirillium) by bacteria of the genus
It is a thermoplastic polyester that is stored as an energy storage material and consists of repeating units of the following formula -CH( _CH3 ) _.CH2.CO.O- . This polymer is conveniently made by culturing microorganisms in an aqueous medium with a suitable substrate such as carbohydrates or methanol as energy and carbon sources. The substrate must, of course, be assimilated by the microorganisms. To promote polymer accumulation, at least a portion of the culturing process is preferably carried out under conditions of limitation of nutrients essential for the growth of the microorganism but not required for the accumulation of the polymer.
Examples of suitable culture methods are described in EP 15669 and EP 46344. Polymers containing both 3-hydroxybutyrate units and other hydroxycarboxylic acid units (eg 3-hydroxyvalerate units) can also be produced by microorganisms. Therefore, heteropolymers containing 3-hydroxybutyrate and 3-hydroxyvalerate residues produced by microorganisms have been classified as "Environmental Science and Technology" by Wallen et al.
Volume 8 (1974), pages 576-579.
Also, as described in EP 52459 and EP 69497, various copolymers are
It can be produced by culturing microorganisms on specific substrates. For example, if propionic acid is used as a substrate,
3-hydroxyvalerate units occur in the copolymer. Therefore, the term "HB polymer" as used herein refers to the homopolymer poly(3
-hydroxybutyrate), but also copolymers such as those mentioned above (provided that the 3-hydroxybutyrate residue also refers to polymers (however, 3-hydroxybutyrate The residues shall constitute at least 40 mol% of the polymer, preferably at least 50 mol%).Although the cells containing the HB polymer can be used as is as a molding material (see e.g. US Pat. No. 3,107,172). , it is usually desirable to separate the polymer from the remaining cellular material. The majority of separation methods proposed to date involve extracting the polymer from the cells with a solvent in which the HB polymer is soluble. The polymer solution (hereinafter referred to as "syrup") is then separated from the cell debris. Typically, such an extraction step involves a treatment that renders the cells permeable to the extraction solvent (e.g., by milling). Typical solvent extraction methods are described in EP 15123. Separation of syrup from cell debris is usually carried out by filtration or centrifugation. about 5% by weight
Syrups containing these polymers tend to be very viscous, making the filtration and centrifugation steps difficult. This is particularly difficult when the proportion of cellular substances to be separated other than the HB polymer (hereinafter referred to as "NPCN") is large. The proportion of NPCM to be separated is, of course, the HB of microbial cells.
Depends on polymer content. Although there have been reports of microbial cells containing high proportions of HB polymers in the literature, there are often practical limits to the proportion of HB polymers in microbial cells due to economic considerations of culture operations, and therefore, the proportion of HB polymers in syrups is limited. The use of large amounts of extraction solvent is required to dilute the syrup sufficiently to facilitate separation of the NPCM from the NPCM. The use of such diluted syrups requires the use of large containers;
Not only are large amounts of solvent recovery costly, but also significant solvent losses are likely to occur (even when relatively efficient solvent recovery operations are used). Therefore, if a syrup concentration of 5% by weight is used, to extract 1Kg of polymer, even if 95% by weight of the solvent is recovered for reuse,
1Kg of solvent is lost. Furthermore, the use of lower concentration syrups and/or less efficient solvent recovery operations results in solvent losses. Furthermore, with lower concentration syrups and/or less efficient solvent recovery operations,
The amount of solvent loss will be even higher. Therefore, in addition to the extraction processing (including solvent recovery) costs, such extraction methods add significant raw material costs (ie, the cost of unrecovered solvent) to the polymer production costs. In the above extraction method, the HB polymer is extracted by dissolution in a solvent, and the NPCN remains undissolved. However, some NPCMs, such as lipids, may also be soluble in the HB polymer extraction solvent and therefore may be present in the syrup. Therefore, if it is desired to obtain an HB polymer product free of such dissolved NPCM impurities, a pre-extraction step with a solvent in which the HB polymer is not soluble removes impurities that are soluble in the HB polymer extraction solvent. Therefore, prior to the extraction of the HB polymer, it is necessary or alternatively the HB polymer must be selectively separated from the syrup, for example by precipitation. Employing selective separation (eg precipitation) steps often further complicates solvent recovery operations. Here, we have devised a method that can separate most of the NPCM from the HB polymer without causing such large losses of solvent and the associated increase in raw material costs. In the present invention, most of the NPCM is solubilized (preferably enzymatically), leaving the HB polymer insoluble. Therefore, in the present invention, an operation opposite to the conventional one is adopted. That is, the NPCM (impurity) is dissolved, leaving the HB polymer in an insoluble state. The dissolution of NPCM can be carried out in several stages so that HB polymer products of increasing purity can be obtained. Of course, the more such stages there are, the higher the operating costs. For some applications of HB polymer products, a lower purity of product than required for other applications can be tolerated, so such a process can be achieved by stepwise extraction of impurities (NPCM). Products of acceptable, lower purity can be produced more cheaply than higher purity products. J. Gen. Microbiology 19 (1958) pages 198-209 proposes separating the polymer from the microbial cells by treating them with an alkaline solution of sodium subzinc chlorate. We have found that although this treatment solubilizes the NPCM, it also causes significant degradation of the HB polymer, making it unsuitable for many applications. There are also many academic publications on HB polymers, such as J. Bacteriology 88 (July 1964), Vol.
On pages 60-71 (particularly page 61), by suspending microbial cells in a lysozyme-containing solution, subjecting the suspension to ultrasound, and then centrifuging the suspension layered on glycerol, A method for the separation of HB polymer granules from microbial cells has been proposed, which consists of separating the HB polymer granules from cell debris. However, such enzymes are not only relatively expensive and their operation is not suitable for large-scale operation;
Only a relatively small proportion of NPCM is solubilized. According to the invention, the NPCM in the HB polymer-containing microbial cells is solubilized by digesting the aqueous suspension of microbial cells with at least one solubilizing agent;
A method for removing NPCM from HB polymer-containing microbial cells, the method comprising: then separating the insoluble residue containing the HB polymer from the suspension; and heating the suspension to a temperature of 80° C. or higher before or during the digestion step and prior to any proteolytic enzyme digestion. The above-mentioned NPCM removal method is provided. Preferably, the heating and digestion steps are carried out sufficiently to solubilize at least 50% by weight of the NPCM in the initial cells and to produce an insoluble residue with an HB polymer content of at least 70% by weight. NPCN usually contains nucleic acids, lipids, phospholipids,
It consists of heptidoglycans, proteinaceous substances (including glycoproteins), and in some cases phosphopolysaccharides, as well as other carbonates. Proteinaceous substances are usually
Consists of at least 40% by weight of NPCM. In the method of the invention, at least some of the above-mentioned NPCM components are solubilized. this is,
This is done by digesting the cells in one or more stages with a solubilizing agent. Preferably, the solubilizing agent in at least one stage is an enzymatic composition, and the digestion step comprises pepsin, trypsin, bromelin, papain, huicin, remin, chymotrypsin, and bacterial or fungal proteolytic enzymes or mixtures thereof. Preferably, the method includes at least one step of treating the suspension with a proteolytic enzyme, such as. A suitable enzyme composition is
It is commonly used in "microbiological" (enzyme-based) detergent powders. The solubilizing agent in at least one digestion step is a proteolytic enzyme and/or a surfactant (especially an anionic surfactant). Before or during the digestion step, but before any proteolytic enzyme digestion step, the cells are subjected to a temperature of 80° C. or higher. Such heating steps cause denaturation and solubilization of some of the nucleic acids in the cells. Omitting such a heating step does not allow satisfactory separation of insoluble residues after the proteolytic enzyme digestion step. This is because, without such a preheating step, the nucleic acids will be released from the cells, giving a very viscous suspension when the cells are digested with proteolytic enzymes. Such nucleic acids could be solubilized by adding deoxyribonuclease prior to treatment with proteolytic enzymes, but high concentrations of relatively expensive deoxyribonuclease are required. Similarly, treatment with a proteolytic enzyme followed by a deoxyribonuclease treatment is impractical due to the difficulty of mixing the deoxyribonuclease with a viscous suspension. If the digestion step involves digestion with proteolytic enzymes, it is preferred to heat the suspension to 100°C or higher, especially at 120°C or higher under a pressure higher than atmospheric pressure sufficient to maintain the aqueous medium in a liquid state. °C or above and then increasing the pressure (e.g. by extruding the heated suspension into a region held at a pressure such that the aqueous medium evaporates).
or by simply cooling the suspension). The heat treatment time required to solubilize and denature nucleic acids will vary depending on the temperature used. The required time is therefore reduced the higher the temperature to which the suspension is applied. Heating for at least 5 minutes, preferably at least 10 minutes,
C., but at higher temperatures, such as 150.degree. C., shorter heating times may be used, sometimes as short as 20 seconds. Any digestion step using a surfactant as a solubility modifier is usually carried out at temperatures above 80°C to provide rapid solubilization by the surfactant. Although a wide range of PH conditions can be used for the heating step to denature and solubilize nucleic acids, the PH conditions should be near neutral, e.g. PH 6-8, to minimize the risk of degradation of the HB polymer. is preferable. In a preferred embodiment of the present invention described above,
At least a portion of the solubilization is accomplished by digestion of the cells with an enzyme composition, particularly by employing at least one digestion step using a proteolytic enzyme composition as a solubilizing agent. Since many enzyme compositions tend to be denatured at temperatures above 60°C, a heating step to denature and solubilize the nucleic acids is performed prior to treatment with the enzyme composition. If an enzymatic digestion step is employed, the digestion should be carried out at a temperature lower than the temperature at which the enzyme is denatured. Denaturation temperature is often 65℃
However, for some enzymes the denaturation temperature is higher and therefore digestion temperatures higher than 65°C may be used when using such enzymes. Preferably, the digestion temperature is below 80°C. Digestion is preferably carried out at temperatures below 60°C, especially between 50 and 60°C, unless the enzyme can withstand temperatures above 60°C. If an enzymatic digestion process is employed, cooling of the cells will usually be required prior to enzymatic digestion, as the suspension is typically heated to a higher temperature than that used in the enzymatic digestion step. . The cells may be separated from the heated suspension, for example by filtration or centrifugation, and then resuspended in another aqueous medium; alternatively, the heated suspension may simply be cooled to the required digestion temperature, whereas Enzymatic digestion steps can also be performed. Solubilized NPCM produced by enzymatic digestion
can be reused, if desired, as part of the substrate for the cultivation of additional quantities of HB polymer-containing microorganisms, thus providing raw material cost savings in the production of HB polymer-containing microorganisms. The enzymatically solubilized NPCM may be recycled to the culture process (after any treatments such as sterilization that may be required). Therefore, solubilization is preferably carried out enzymatically. Further solubilization of NPCM in the residue remaining after enzymatic digestion can be carried out using surfactants as solubilizing agents. When solubilization is carried out using both a proteolytic enzyme composition and a surfactant as solubilizing agents, the digestion is carried out in several stages, with the surfactant digestion stage being replaced by the enzyme digestion stage(s). It is preferable to carry out after. There are two reasons for this; firstly, the enzyme composition can be inactivated by surfactants, and secondly, solubilized NPCM is added to the culture process used to create the microbial suspension. If recirculation is desired, the presence of surfactants in the solubilized portion of the suspension may preclude such reuse. To achieve sufficient or better solubilization of NPCM, the digestion step is usually
A digestion step using a phospholipase enzyme may be included to solubilize the phospholipids, which make up about 6-10% by weight of the NPCM. Enzymatic digestion is preferably carried out by maintaining the cell suspension to which the enzyme composition has been added at the required temperature and pH in the range of 6.5 to 9.0 until the desired degree of processing is achieved, for a period of time , usually about 0.2 to 2 hours. Enzymatic digestion may be carried out in several stages, for example, a first stage may use one enzyme composition, followed by one or more processing stages using the same or different enzyme compositions. However, if more than one enzyme is used, it may be convenient to treat the cells in a single step with one enzyme mixture. In fact, we have shown that in some cases using enzyme mixtures can have a synergistic effect, so that if the enzymes do not digest each other, in some cases using enzyme mixtures can cause enzymes to be used alone. It has been discovered that use of NPCM in combination or sequentially results in a higher degree of solubilization of NPCM. The required amount of enzymes, such as protease and/or phospholipase enzymes, will depend on the type and activity of the enzyme, typically the amount of protease will initially range from 0.5 to 100 g of NPCM in the cell.
The amount is such as to give 10 Anson units (AU), preferably 1 to 6 AU. Proteolytic enzyme activity can be measured by enzymatically digesting denatured hemoglobin for 10 minutes at 25° C. and PH 7.5. 1AU is the amount of phenolic reagent that gives the same color as 1 milliequivalent of tyrosine.
TCA soluble product per minute (at initial rate)
This is the amount of enzyme released. A detailed description of this analytical method is available from Novo Industries.
Industries) published leaflet AF4. Although certain enzymes, such as lysotium, solubilize peptidoglycan, we
It has been discovered that it is desirable to use enzyme compositions that result in little or no solubilization of peptidoglycan in NPCM, at least during the initial digestion steps. The reason for this is that if the peptidoglycan is solubilized, separation of the insoluble HB polymer granules from the solubilized material tends to be more difficult. It is thought that the peptidoglycan then forms some sort of net or pouch that envelops the agglomerates of HB polymer granules. Such agglomerates can be separated from the aqueous medium more easily than deagglomerated granules.
The presence of peptidoglycan can be confirmed by measuring the diaminopimelic acid content of the insoluble residue containing the HB polymer. As mentioned above, solubilization of NPCM can also be achieved by using detergents as solubilizing agents, which is preferably carried out after the proteolytic enzyme digestion step. Solubilization with surfactants, e.g. anionic surfactants such as sulfated or sulfonated fatty acids (especially sodium dodecyl sulfate), can be achieved by heating the surfactant-added suspension at temperatures above 80°C. Preferably, this is carried out by heating to . Preferably, the amount of surfactant used is 10-20% by weight of the NPCM remaining in the suspension. Adding complexing agents such as ethylenediaminetetraacetic acid to surfactants
It may be advantageous in promoting solubilization of NPCM. In some cases where surfactant treatment is carried out after enzymatic digestion, we recommend that such surfactant It has been discovered that emulsions can be formed during processing and that solids are very difficult to separate from such emulsions. We have discovered that the addition of cationic flocculants or electrolytes to such emulsions is not very effective in promoting their separation. However, acidification to pH below 2 or addition of absorbent minerals such as diatomaceous earth promotes separation. However, acidification may cause some precipitation of surfactant-solubilized NPCM. The insoluble residue remaining after the digestion step contains some residual undissolved NPCM along with the HB polymer. The degree of solubilization of NPCM at any given stage can be conveniently calculated from the initial cells and the HB polymer-containing compositions obtained at that stage and their respective HB polymer contents. For the purpose of this calculation, assume that there is no loss and that no HB polymer was solubilized. Thus, if the initial cells contain HB polymers with P ₀ % by weight and the product at that stage has an HB polymer content of ₁ % P by weight, the solubilized NPCM
The percentage N of is given by the following formula. N=[1- _P0 (100- _P1 )/ _P1 (100- _P0 )]×10
0 The heat treatment and digestion steps are preferably carried out such that at least 50% by weight of the NPCM of the initial cells is solubilized and the residue comprises at least 70% by weight, preferably at least 85% by weight of HB polymer. The amount of digestion required to achieve this is
Of course, it will depend on the initial HB polymer content P ₀ of the microbial cells. Preferably, the microorganism is cultured under conditions such that the HB polymer content P ₀ is at least 50% by weight. However, as mentioned above, due to economic considerations, the cell's HB polymer content P ₀ is 80
It may be limited to % by weight or less. In a preferred embodiment of the invention, after solubilization of the NPCM by enzymes and/or surfactants, the residue is treated with hydrogen peroxide. proteinaceous
If most of the NPCM has been solubilized with proteolytic enzymes, treatment with hydrogen peroxide will have little or no effect on the further solubilization of residual NPCM and will reduce the amount of HB polymer-containing residue. Desirable for removing stains.
Treatment with hydrogen peroxide may provide an advantage by allowing the HB polymer-containing residue to be more easily separated (eg, by filtration) from the aqueous medium. In other cases (e.g. when proteolytic enzyme digestion is used to solubilize only a portion of the proteinaceous NPCM and/or when surfactant digestion is employed), hydrogen peroxide treatment , additional amounts of NPCM may be removed. If the NPCM of the HB polymer-containing residue contains lipids, e.g. if digestion with a phosphoryhase enzyme was not carried out, the HB polymer-containing residue can be treated with a solvent that dissolves the lipids but not the HB polymer, e.g. methanol. Lipids can be removed by washing. Such a solvent cleaning step may be desirable as a deodorizing step. The above operation yields an insoluble residue,
This usually contains at least 70% by weight, preferably at least 85% by weight, especially at least 90% by weight.
Contains HB polymer. Preferably, no significant solubilization of the peptidoglycan is carried out, as mentioned above. Preferred products therefore contain at least 90% by weight of HB polymer and at least 1% by weight, especially from 1 to 3% by weight of heptidoglycan. Preferably the content of proteinaceous substances in such products is less than 6% by weight. In some cases, the product from the digestion process can be used as such, for example as a molding material. Alternatively, the HB polymer can be used as a solvent for the HB polymer;
Especially methylene chloride, chloroform, or 1,2
- Can be extracted by solvent extraction with halogenated hydrocarbons such as dichloroethane. Since the residual proportion of NPCM is small, the separation of NPCM from the syrup, e.g. by filtration, is much easier than in direct solvent extraction methods, and therefore even thicker syrups, e.g. syrups containing 5-15% by weight of polymer, are much easier to separate from the syrup than in direct solvent extraction methods. can be adopted. Therefore, the required proportion of extraction solvent can be reduced and thus solvent losses due to incomplete solvent recovery can be reduced. EXAMPLES The invention is further illustrated in the following examples. All percentages in the examples are given on a weight basis. The suspensions used in Examples 1 to 5, 7 to 16, and 18 to 19 were derived from Alcalignes eutrophus grown in continuous culture under nitrogen limitation using glucose as a substrate.
11599) by centrifugation of the culture solution. Example 1 In this example, cells with approximately 60% 3-hydroxybutyrate homopolymer (PHB) content were
Several suspensions containing g/g/g were heated at 100°C.
The mixture was boiled under reflux conditions for various times. The suspensions were then cooled and tris(hydroxymethyl)aminomethane hydrochloride was added as a buffer to give a buffer concentration of 50mM.
These buffered suspensions were then incubated for 1 hour at 55° C. and PH 8.2 with a 1% protease composition based on initial cell dry weight. The protease composition used here was "Alcalase (trademark) 0.6L" obtained from Novo Industries, and according to sales literature published by the company, it has an activity of 0.6 AU/g. has been done. This amount of "alcalase" corresponds to approximately 1.5 AU/100 g of NPCM in the initial cells. A sample of the resulting digestive suspension was diluted with ice-cold water and then centrifuged at 20000G for 1-2 minutes. The resulting centrifuged pellets were washed three times with water and their protein content was then determined by J.Biol.Chem.
It was measured by a method based on the colorimetric analysis method published by Lowry et al., 193 (1951) 265. The results are shown below. [Table] When compared with the protein analysis values obtained using the amino acid analysis method, it was found that the colorimetric analysis method provides values that are approximately 75% of those obtained using the amino acid analysis method. Example 2 The effectiveness of the heat treatment step can also be assessed by measuring the sedimentation rate of the suspension after heat treatment and before any enzyme treatment. The aqueous suspension used contained cells containing 72% PHB at a concentration of 20 g/cell. The suspension was heat treated by heating in an autoclave for 5 minutes and then centrifuged in a strobe centrifuge at 100 rpm. The height of the solid/liquid interface was measured after various centrifugation times. [Table] It can be seen that the higher the temperature, the easier the separation takes place. Example 3 The operation of Example 1 was repeated, but in this example
A suspension containing 50 g/cells with a PHB content of 52% was used. Boiling was carried out for 10 minutes. To remove phospholipids, after digestion with "Alcalase", the suspension was incubated for 1 hour at 40° C. and PH 8.6 with the addition of various amounts of phospholipase enzyme composition.
The phospholipase enzyme composition was "Lecitase 100S" from Novo Industrieis. By measuring the protein content of the product before and after "recitase" treatment as in Example 1, and by extracting the phospholipids with a solvent and assaying the extraction by an enzymatic method,
The residual phospholipid content was determined. The enzymatic assay used phospholipase C enzyme. Since this hydrolyzes phosphatides other than phosphatidylethanolamine relatively slowly, only about 50% of the phospholipids present are detected with this method. Therefore, in the table below, phosphatidylethanolamine content is indicated rather than total phospholipids. For comparison, the above operation was performed using "Alcalase"
Digestion was omitted and repeated. [Table] * Weight % based on initial cell dry weight
※※ PHB of products and initial cells
Calculated from the content Although lower levels of phosphatidylethanolamine can be achieved, heat treatment and “recitase”
It can be seen that digestion alone cannot solubilize enough NPCM to give a product containing more than 70% polymer, even when relatively large amounts of "recitase" are used. However, if the "recitase" treatment is performed after the "alcalase" treatment, a significant improvement in polymer purity can be achieved, with "recitase" affecting the solubilization of only small amounts of protein. Example 4 This example compares various concentrations of "Alcalase 0.6L" protease and various digestion times. The procedure of Example 1 was repeated. In this example, the boiling time was 1 hour, and then digestion was performed with 0.6 L of "Alcalase" of various concentrations. Samples were taken at regular intervals from each digestion treatment and analyzed for protein using a colorimetric method. The table below shows the percentage of protein in the initial cells that was solubilized, rather than the actual protein content of the product. [Table] [Table] *Based on initial cell weight.
Example 5 In this example, the effectiveness of surfactant digestion is evaluated. The cell suspension used was the same as in Example 3. The following process was adopted. A. Boil at 100℃ for 1 hour under reflux conditions. B. Addition of 10% dodecyl sulfate sodium salt based on cell dry weight followed by boiling at 100° C. for 1 hour under reflux conditions. CB Same as B, but add enough ethylenediaminetetraacetic acid to give a 5mM solution before boiling. DA Same as A, then add 0.6L of 1% "Alcalase" based on cell dry weight and digest at 55℃, pH 8.2 for 1 hour. ED Same as D, then perform B. FD Same as D, then perform C. In this example, protein content was measured by amino acid analysis. The diaminopimelic acid content gives an indication of the concentration of residual peptidoglycan. The peptidoglycan content is approximately 5 times the stated diaminopimelic acid content. TABLE It can be seen that although the various treatments described above gave significantly reduced protein concentrations, the diaminopimelic acid content indicates that little peptidoglycan was solubilized. In another experiment, cells with 60% PHB content were
When a suspension containing 50 g/concentration was subjected to the above treatment F, similar results were obtained. Further digestion with "Alcalase" or dodecyl sulfate sodium salt after treatment F did not significantly improve the purity of the product. Example 6 In this example, samples were obtained by centrifuging a culture of Alcaligenes eutrophus (NCIB Deposit No. 11599) grown in continuous culture under nitrogen limitation using a mixture of glucose and propionic acid as substrate. In addition, 3-hydroxybutyrate/3-hydroxyvalerate copolymer (containing 10 mol% 3-hydroxyvalerate units) was added to 48
A suspension containing 50 g/% of the microbial cells was used. Process F of Example 5 was repeated, but this time the initial boiling before the "Alcalase" digestion was for 10 minutes instead of 1 hour. After digestion with dodecyl sulfate sodium salt, the product was divided into several parts and centrifuged to obtain pellets. Add one pellet to 50mM phosphate buffer (already
EDTA was added at a concentration of 1 mM). 0.1% egg white lysozyme (Sigma Chemicals) based on pellet weight
Chemicals) was added thereto, and the suspension was digested at 20°C and pH 6.5 for 1 hour. Another pellet was suspended in 50mM acetate buffer supplemented with 1mM EDTA and added with 0.1% Novozyme 234 (Novo Industrieis) based on the weight of the pellet. and the suspension was digested for 1 hour at 50°C and PH4.5. Another pellet was digested in 0.1M aqueous sodium hydroxide for 1 hour at 20°C. This alkaline digestion treatment was also applied to the respective products obtained from the lysozyme and "novozyme" digestions described above. [Table] *Based on amino acid analysis.
It can be seen that lysozyme significantly reduced the diaminopimelic acid content. Although alkaline treatment and “Novozyme” did not have a significant effect on the content of diaminopimelic acid, the purity of the HB polymer was improved when alkaline treatment was employed alone or when alkaline treatment was employed after lysozyme. Examples 7-11 In these examples the suspensions had a PHB content of 79%.
of cells at a concentration of 90g/. The suspension was heat treated in an autoclave by blowing in pressurized steam and heating to 14° C. for 3 minutes. The suspension was then cooled and centrifuged. Aliquots of the solid residue were resuspended in water to make suspensions with a solids content of 50 g/ml. Various commercially available enzyme compositions are added to these suspensions to bring the suspensions to 50% pH at the PH value recommended by the respective enzyme composition supplier (adjusted if necessary by the addition of sodium hydroxide). Digested for 60 min at °C. The inert residue was then separated from the aqueous medium by centrifugation and washed with deionized water. The results are shown in the table below. Table: The NPCM in the products of Examples 9 and 10 could be further solubilized by further subjecting the residue to enzymatic and/or surfactant digestion. Comparing Example 11 with Examples 9 and 10 shows that using a mixture of proteolytic enzymes is superior to using equivalent amounts of individual enzymes.
It can be seen that NPCM can be solubilized. Example 12 The product of Example 11 was resuspended in water. The above suspension was digested for 60 minutes at 50° C. and pH 7.0 using 0.25 g of “Neutrase 0.5 L” in combination with 0.25 g of “Alcalase 0.6 L”. The residue was separated from the aqueous medium by centrifugation and washed with deionized water. The residue is 96
Contained % PHB. This is 84% overall
Corresponds to NPCM solubilization. Examples 13-16 The operations of Examples 7-11 were repeated, but the PHB content
A 75% cell suspension was used, various proteolytic enzyme compositions were used, and the digestion process was carried out at the temperature recommended by the respective enzyme composition supplier.
The experiment was carried out at pH 7 for 60 minutes. The amount of enzyme composition used in each example was 1% based on initial cell dry weight. [Table] Bromelain concentrate (active
1295BTU/g; obtained from pineapple trunk)
and papain (30000 PU/mg active; obtained from papaya fruit) were both supplied by Miles Takamine, El Hart, Indiana, USA. "Allprotease"
and "High T" were supplied by All-Tech, Lexington, Ky., USA. "All protease"
is a mixture of fungal, bacterial and plant enzymes, while "High-T" is derived from Bacillus licheniformis (B.
licheniformis). Example 17 The suspension used in this example consisted of cells of Methlobacterium organophilum (NCIB Deposit No. 11483) obtained by batch culture under nitrogen limitation using methanol as a substrate. It was a suspension. Cells are 17%
contained PHB. The operations in Examples 7 to 16 were repeated, but in this example, a mixture of 0.5% "Alcalase 0.6L" and 0.5% "Neutrase 0.5L" (each percentage based on the initial cell dry weight) was added to the enzyme composition. Digestion time was 60 minutes, pH was 7.0, and temperature was 55°C.
The product was further subjected to multiple digestion steps. using the same conditions, using new enzyme for each digestion, and centrifuging the product between digestion steps,
Resuspend in deionized water. The results were as follows. [Table] Example 18 The suspension used in this example had a PHB content of 5%.
cells at a concentration of 100g/. 500 ml of the suspension was charged to the first stirred autoclave. The same amount of water was charged to a second stirred autoclave and heated to 350°C under nitrogen pressure. the first autoclave is pressurized with nitrogen to a pressure that exceeds the pressure in the second autoclave;
The contents of the first autoclave were then forced into the second autoclave by virtue of its excess nitrogen pressure. The combined contents of the second autoclave are
Mixed vigorously for an hour. The temperature of the combined contents in the second autoclave was approximately 170°C, but the applied pressure was sufficient to maintain a liquid state. The contents of the second autoclave were then forced by nitrogen pressure into a collection vessel at atmospheric pressure. The resulting product was centrifuged, the centrifuged pellet was resuspended in 500 ml of deionized water, and then 0.5 g of "Alcalase 0.6 L" was added to the suspension. The suspension was kept at 55°C and PH7 for 30 minutes. The suspension was then centrifuged at 5000G for 10 minutes. The centrifuged pellet was then resuspended in 500 ml of deionized water. 5g, i.e. residual
Approximately 107% of NPCM sodium dodecyl sulfate salt was added and the suspension was heated at 100° C. for 1 hour. The suspension was then centrifuged at 5000G for 10 minutes to obtain a pellet, which was washed twice with deionized water (the pellet was collected by centrifugation between the washes). Finally, the dried brown product (Product A)
I got it. I repeated the above operation, but "Alcalase"
The digestion step and the "recitase" digestion step were carried out in combination, using a mixture of 0.5 g "alcalase" and 0.5 g "recitase" for the digestion. [Table] If the "Alcalase" digestion treatment is carried out directly on the initial suspension (i.e. without the heat treatment step), the suspension becomes extremely viscous and cannot be stirred, or insoluble parts No treatment could be done to separate them. A portion of the brown product A from above was heated under reflux conditions.
Extracted with 10 parts of methylene chloride and filtered the resulting syrup. Despite the high viscosity of this syrup, standard syrup filtration methods such as those employed in cellulose triacetate film production could be used. The reason was that the proportion of NPCM to be removed was only about 0.5% of the syrup. Later on, Shirotsupu was able to make PHB films as a cast member. Adding some of the syrup to petroleum ether, HB
The polymer was precipitated to obtain a fine white powder (B). Samples of each of Product A and Powder B were melt extruded in the following manner: A 3.5 g sample was extruded on a melt flow grader (Daven Test, Welwyn, UK) equipped with a die with a circular orifice of 2 mm diameter and 8 mm land length. It was placed in a barrel manufactured by the company. The barrel was maintained at 190°C. 10 after a 5 minute warm-up period
A load of Kg is applied to the piston (the weight of the piston
0.16Kg). Melt flow time is the total time required for a total of 2 g of sample to be extruded through the die (including the 5 minute warm-up time mentioned above). The melt flow times were as follows. Product A 10.5 min Powder B 8.0 min For comparison, the PHB sample isolated from Alcaligenes eutrophus cells by the spray drying/liquid extraction/solvent extraction method as described in EP 15123. Melt flow times typically range from 8 to 10 minutes. This indicates that the melt stability of the polymer either before or after the methylene chloride extraction step described above is comparable to that of the polymer extracted by the direct solvent extraction route. Product A had a weight average molecular weight of approximately 1,000,000 as determined by gel permeation chromatography. For comparison, PHB was extracted from the same suspension used as starting material in this example by digestion with sodium subzinc chloride (15% based on cell dry weight) at 40°C for 30 min. In some cases, a product with a weight average molecular weight of only about 101,000 was obtained. This indicates that digestion with perchlorate results in significant degradation of the polymer. Example 19 The suspension used in this example was the same as that used in Example 18. 500ml of the suspension was heated to 80°C by a steam heating coil immersed therein and then cooled to 55°C. The treatments described in Example 18 with "Alcalase", "Lecitase" and dodecyl sulfate sodium salt were carried out in this order. The suspension became slightly viscous after "Alcalase" digestion, but was still stirrable.
However, the centrifugation steps after each of the "alcalase" and "recitase" digestions were more difficult. The reason for this was that the floc was extremely delicate, and unlike the hard pellets obtained in Example 18, gel-like pellets were obtained. However, after treatment with dodecyl sulfate sodium salt, a stable suspension was formed that could be easily separated by centrifugation. To investigate the sedimentation properties of this suspension, 20 ml of it was placed in a test tube to provide a vertical column of suspension with a height of 6 cm. Various additives were added to the suspension and the height of the solid/liquid interface was observed after various times. The results are shown in the table below. [Table] −= No interface Electrolyte (CaCl ₂ ) or cationic flocculant (“Aquaflock” 4051 or
4067) has no effect, but diatomaceous earth or large amounts
It can be seen that addition of HCl can lower the pH below 2 and cause precipitation. Centrifugation at low gravitational acceleration (G) of columns of similar suspensions also removes undenatured suspensions, and pH with HCl.
It was carried out on suspensions acidified to 1.60. The height of the interface after various centrifugation times at 500G is shown in the table below. [Table] Example 20 Alcaligenes eutrophus (NICB Deposit No.
11599) in an aqueous medium using a mixture of glucose and propionic acid as a substrate under nitrogen conditions.
A culture solution was obtained containing cells containing 71% hydroxyvalerate (HV) copolymer (HB:HV molar ratio = 4.9:1) at a concentration of 21 g/cell. The culture solution is transferred from the incubator through a sterilizer maintained at 135°C and then through a cooler at 70°C to 130°C.
into the storage vessel at a flow rate of Residence time in the sterilizer was approximately 7 minutes. The pH of the culture solution in the storage container was adjusted to 8, and then 0.2 g/0.6 L of "Alcalase" was added when the temperature dropped to 50°C. The mixture was left in a storage container overnight. Meanwhile the temperature of the mixture is 27
The temperature dropped to ℃. The mixture was then centrifuged. The HB copolymer content of the solid residue was 84%, which corresponds to 53% solubilization of NPCM.
This residue was resuspended in water to a solids content of 20 g/
A suspension was made and this suspension was then split into two parts and processed separately. A Add 0.2g/alcalase to the first part.
0.6 L'' and the mixture was digested at 55°C and PH8 for 1 hour, then centrifuged. The HB copolymer content of the residue is 92%, which is
This corresponded to 79% solubilization of NPCM. This residue was then suspended in water to yield approximately 260g/
with a solid content of 10 vol% hydrogen peroxide 500
ml/ for 2 hours at 80°C. This decolorized residue was separated by centrifugation, washed with water, and dried. The HB copolymer content of this residue was 92%. This is due to hydrogen peroxide treatment.
This shows that NPCM was not further solubilized. B. To the second part of the two-part suspension was added 2 g/d of dodecyl sulfate sodium salt and the mixture was heated to the boil for 1 hour. The mixture was then centrifuged to obtain a residue with 87% HB copolymer content. This value corresponds to 63% solubilization of NPCM. This residue was divided into two parts and processed separately. The first part of the residue was suspended in water and weighed approximately 190g/
A suspension with a solid content of 10% was prepared and digested at 80°C for 2 hours using 500ml/10vol% hydrogen peroxide. The decolorization residue was separated by centrifugation, washed with water, and dried. The HB copolymer content of this dried residue was 96%, which corresponded to a total solubilization of 90% of NPCM. A second portion of the residue was washed with methanol, then water, and then separated by centrifugation. The HB copolymer content of this residue is 93%, which is
This corresponded to 82% solubilization of NPCM. This wet residue was resuspended in water to give 200g/
500% solids content and 10vol% hydrogen peroxide.
ml/ml for 2 hours at 80°C. The resulting decolorized slurry was centrifuged and
A residue was obtained which was then washed with water and dried. The HB copolymer content of this dry residue is 98%
This corresponded to 95% solubilization of NPCM. Example 21 Alcaligenes eutrophus (NCIB Deposit No.
11599) in an aqueous medium under nitrogen limitation using a mixture of glucose and propionic acid as substrate to obtain 45 g of cells containing 75% HB/HV copolymer (HB:HV molar ratio = 4:1). A suspension containing a concentration of / was obtained. (In this example, the amounts of enzyme and surfactant are expressed as percentages based on the initial cell dry weight.) The suspension was passed through a pipe and heated to 150°C by blowing steam through the pipe. The residence time at 150°C was 20 seconds. The resulting suspension
Cool to 70℃ and add 0.5% "Alcalase 0.6L" and 0.5% "Neutrase" at that temperature.
Digestion was performed using 0.5 L of the mixture at pH 7.5 for 2 hours. The resulting suspension was concentrated by centrifugation, then 3% dodecyl sulfate sodium salt was added,
It was boiled at 100° C. for 2 hours under reflux conditions. The resulting suspension was then spray dried. The spray-dried powder was washed with methanol under reflux conditions, filtered and dried. At various stages mentioned above
The HB polymer content is shown in the table below. [Table] Calculated from mer content.

Claims (1)

[Scope of Claims] 1. Digesting an aqueous suspension of microbial cells containing 3-hydroxybutyrate polymer with at least one solubilizing agent allows cell material other than 3-hydroxybutyrate polymer to be released from the cells. A method for removing cellular materials other than 3-hydroxybutyrate polymer from microbial cells containing 3-hydroxybutyrate polymer, comprising solubilizing and then separating the insoluble residue containing the 3-hydroxybutyrate polymer from the suspension. and: () the digestion step includes one or more steps of using a proteolytic enzyme composition as a solubilizing agent, and the aqueous suspension is subjected to the proteolysis before or during the digestion step; (a) the digestion step also includes at least one step using phospholipase as a solubilizing agent; or (b) the enzyme and treating the insoluble residue with hydrogen peroxide after digestion with hydrogen peroxide.